Despite the advent of highly active antiretroviral therapy (HAART), AIDS continues to be a major health problem. There are currently an estimated 30 to 40 million cases of HIV worldwide and projections indicate that if the pandemic proceeds there will be 45 million new infections by 2010 and nearly 70 million deaths by 2020. While HAART has substantially reduced the morbidity and mortality associated with HIV infection, emergence of resistant viral variants, toxicity and other compliance or availability issues have presented significant obstacles in controlling the disease. Thus alternative treatment strategies will be required. Anti-HIV gene therapy strategies based on RNAi technology have been proposed using T cells or hematopoietic stem cells (HSCs). We propose to genetically modify HSCs to protect all hematopoietic progeny including T lymphocytes and macrophages from HIV infection. Unfortunately murine or in vitro models have not been predictive for stem cell transduction in humans. Results in large animal models, in particular nonhuman primates, have correlated much better with human studies. We have successfully used the nonhuman primate model to develop efficient stem cell transduction strategies. In the past we have shown efficient oncoretroviral gene transfer to long-term repopulating cells in nonhuman primates after myeloablative conditioning. Unfortunately, oncoretroviral gene transfer protocols require prolonged transduction cultures for efficient stem cell transduction, which will in turn lead to a significant loss of stem cells. While this may be acceptable in a myeloablative setting in which endogenous stem cells are maximally depleted, this approach will most likely not lead to efficient gene !transfer in a nonmyeloablative setting, likely necessary for the treatment of AIDS patients, because a significant number of endogenous stem cells will survive the conditioning regimen and be able to compete with the transduced stem cells. Thus, we propose to use lentiviral vectors to significantly shorten the transduction culture, which should minimize stem cell loss and therefore maximize the ability to compete with endogenous stem cells and thereby improve engraftment. We propose to 1) develop nonmyeloablative strategies to achieve engraftment of lentivirally modified stem cells, 2) develop in vivo selection strategies to further expand lentivirally modified cells in nonhuman primates, and 3) test whether these strategies will allow for efficient protection of hematopoietic cells from HIV/SHIV infection. Performing these studies in a clinically highly relevant nonhuman primate AIDS model will allow us to generate the necessary preclinical safety and efficacy data for future clinical anti-HIV studies using lentivirally transduced stem cells.